primary human cd4 t lymphocytes  (Miltenyi Biotec)

Journal: Cell Cycle

Article Title: Exogenous expression of SAMHD1 inhibits proliferation and induces apoptosis in cutaneous T-cell lymphoma-derived HuT78 cells

doi: 10.1080/15384101.2016.1261226

Figure Lengend Snippet: Exogenous SAMHD1 expression inhibits HuT78 cell growth and proliferation. HuT78 cells expressing hemagglutinin (HA)-tagged SAMHD1 were generated via lentiviral vector-mediated transduction. (A) Exogenous expression of SAMHD1 was validated by immunoblotting analysis using antibodies to HA (SAMHD1) and GAPDH (loading control). (B) Total RNA was isolated from HuT78 vector or SAMHD1-expressing cells and qPCR analysis was performed to quantify the relative SAMHD1 mRNA levels. GAPDH mRNA levels were quantified as internal control. (C) HuT78 vector control and SAMHD1 expressing cells were seeded in triplicate in 24-well plates at a density of 1 × 104 cells/well in 1 ml culture media on day 0 and live cells were counted on indicated days via trypan-blue exclusion method. (D) Cell lines were seeded on day 0 in 4 replicates in 96-well plates at density of 1 × 103 cells/well in 100 µl and cell proliferation was determined on indicated days utilizing an MTS-based proliferation assay. All the data presented are representative of 3 or more independent experiments. (C-D) ***, p < 0.0001.

Article Snippet: Human CD4 + T-lymphocytic cell line HuT78 derived from an Sézary syndrome patient was obtained from the American Type Culture Collection (ATCC, TIB-161).

Techniques: Expressing, Generated, Plasmid Preparation, Transduction, Western Blot, Control, Isolation, Proliferation Assay

Journal: Cell Cycle

Article Title: Exogenous expression of SAMHD1 inhibits proliferation and induces apoptosis in cutaneous T-cell lymphoma-derived HuT78 cells

doi: 10.1080/15384101.2016.1261226

Figure Lengend Snippet: Exogenous SAMHD1 expression in HuT78 cells results in reduced colony formation. HuT78 vector control, and SAMHD1-expressing cells were plated in duplicate in 6-well plates at a density of 1 × 103 cells/well in 1 ml methyl cellulose medium and were left in culture for 10 d. (A) Numbers of colonies were counted after 10 d following standard counting criteria (all colonies with >25 cells were counted). Data is presented as % of colony yield relative to vector control cells (left panel). **, p < 0.005, Representative images (20 × magnification) of colonies formed after 10 d (right panel). (B) Numbers of colonies of different sizes were counted after 10 d based on following standard counting criteria (small: <25 cells; medium: 25–50 cells, and large: >50 cells). Data is presented as percentages of colony yield relative to vector control cells. All the data presented are representative of 3 independent experiments.

Article Snippet: Human CD4 + T-lymphocytic cell line HuT78 derived from an Sézary syndrome patient was obtained from the American Type Culture Collection (ATCC, TIB-161).

Techniques: Expressing, Plasmid Preparation, Control

Journal: Cell Cycle

Article Title: Exogenous expression of SAMHD1 inhibits proliferation and induces apoptosis in cutaneous T-cell lymphoma-derived HuT78 cells

doi: 10.1080/15384101.2016.1261226

Figure Lengend Snippet: Exogenous SAMHD1 expression induces apoptosis and caspase-3/7 activity via activation of extrinsic apoptosis signaling. HuT78 vector control and SAMHD1 expressing cells were seeded at density of 2 × 104 cells per 6-cm dish containing 5 ml growth media on day 0 following trypan-blue exclusion method. (A) Seven days post-seeding, cells in triplicate, were stained with propidium iodide (PI) and cell cycle analysis was performed via flow cytometry. Percentages of cells in G1/G0, S, and G2/M phases of cell cycle are presented. (B) Percentage of cells in Sub-G1 phase are presented. (C) HuT78 vector control and SAMHD1-expressing cells were seeded at density of 2 × 104 cells per 6-cm dish containing 5 ml growth media on day 0 following trypan-blue exclusion method. Seven days post-seeding, cells were stained with annexin V-PE and 7-AAD in triplicate and analyzed by flow cytometry. Representative flow cytometric profiles are presented (left panel). The percentages of non-apoptotic cells, early apoptotic cells, and late apoptotic cells were quantified as presented (right panel). (D) On day 7 post-seeding, cell lysates from all cell lines were collected and western analysis was performed using the antibodies to caspase-8, caspase-9, caspase-3, PARP, HA (SAMHD1), and GAPDH (loading control). (E) On day 7 post-seeding, 1 × 104 cells per cell line were collected in 4 replicates and incubated in 100 µl of Caspase-Glo 3/7 reagent at room temperature in dark for 1 hour. Caspase-3/7 activity was then determined by measuring luminescence values. All the data presented are representative of 3 independent experiments. B, C, E, ***, p < 0.001.

Article Snippet: Human CD4 + T-lymphocytic cell line HuT78 derived from an Sézary syndrome patient was obtained from the American Type Culture Collection (ATCC, TIB-161).

Techniques: Expressing, Activity Assay, Activation Assay, Plasmid Preparation, Control, Staining, Cell Cycle Assay, Flow Cytometry, Western Blot, Incubation

Journal: Cell Cycle

Article Title: Exogenous expression of SAMHD1 inhibits proliferation and induces apoptosis in cutaneous T-cell lymphoma-derived HuT78 cells

doi: 10.1080/15384101.2016.1261226

Figure Lengend Snippet: Exogenous SAMHD1 expression in HuT78 cells significantly increases Fas-L induced apoptosis and caspase-3/7 activity. (A) HuT78 vector control and SAMHD1-expressing cells were treated with 100 ng/ml Fas-L for 48 hours. After 48 hours of treatment, all the cells in triplicate were stained with annexin V-PE and 7-AminoactinomycinD (7-AAD) followed by flow cytometry. Representative flow cytometric profiles are presented (left panel). The percentage of non-apoptotic cells, early apoptotic cells, and late apoptotic cells were quantified as presented (right panel). (B) HuT78 vector control and SAMHD1-expressing cells were treated with 100 ng/ml Fas-L for 48 hours. Post-treatment, 1 × 104 cells per cell line were collected in triplicate and incubated in 100 µl of Caspase-Glo 3/7 reagent at room temperature in dark for 1 hour. Caspase-3/7 activity was then determined by measuring luminescence values. C, HuT78 vector control and SAMHD1-expressing cells were treated with Fas-L (100 ng/ml) for 48 hours. Post-treatment, cell lysates from all cell lines were collected and immunoblotting was performed using the antibodies to caspase-8, caspase-3, PARP, and GAPDH (loading control). All the data presented are representative of 3 independent experiments. A–B, ***, p < 0.001.

Article Snippet: Human CD4 + T-lymphocytic cell line HuT78 derived from an Sézary syndrome patient was obtained from the American Type Culture Collection (ATCC, TIB-161).

Techniques: Expressing, Activity Assay, Plasmid Preparation, Control, Staining, Flow Cytometry, Incubation, Western Blot

Journal: Cell Cycle

Article Title: Exogenous expression of SAMHD1 inhibits proliferation and induces apoptosis in cutaneous T-cell lymphoma-derived HuT78 cells

doi: 10.1080/15384101.2016.1261226

Figure Lengend Snippet: Exogenous SAMHD1 expression in HuT78 cells reduces the levels of cFLIPS protein and mRNA. HuT78 vector control and SAMHD1-expressing cells were seeded at density of 2 × 104 cells per 6-cm dish containing 5 ml growth media on day 0 following trypan-blue exclusion method. (A) On day 7 post-seeding, cell lysates were collected and immunoblotting analysis was performed for cFLIP, HA (SAMHD1) and GAPDH (loading control) (left panel). Densitometry was performed using ImageJ to quantify the relative cFLIPL and cFLIPS protein levels (right panel). ***, p < 0.001 (B) On day 7 post-seeding, total RNA was isolated from HuT78 vector or SAMHD1-expressing cells and qPCR analysis was performed to quantify the relative cFLIPL and cFLIPS mRNA levels. GAPDH mRNA levels were quantified as internal control. All the data presented are representative of 3 independent experiments. *, p < 0.05.

Article Snippet: Human CD4 + T-lymphocytic cell line HuT78 derived from an Sézary syndrome patient was obtained from the American Type Culture Collection (ATCC, TIB-161).

Techniques: Expressing, Plasmid Preparation, Control, Western Blot, Isolation

Journal: Cell Cycle

Article Title: Exogenous expression of SAMHD1 inhibits proliferation and induces apoptosis in cutaneous T-cell lymphoma-derived HuT78 cells

doi: 10.1080/15384101.2016.1261226

Figure Lengend Snippet: Summary of the findings and proposed mechanisms. Our data indicate that increased SAMHD1 expression inhibits growth and proliferation in CTCL-derived CD4+ T-cells by inducing spontaneous and Fas-L stimulated apoptosis. SAMHD1 expression also diminishes colony forming potential in these cells. Furthermore, increased SAMHD1 expression also significantly reduces the mRNA and protein levels of cFLIPS, a key anti-apoptotic protein. Complete mechanisms by which SAMHD1 regulates cFLIPS expression, apoptosis and colony formation in HuT78 cells remain to be understood.

Article Snippet: Human CD4 + T-lymphocytic cell line HuT78 derived from an Sézary syndrome patient was obtained from the American Type Culture Collection (ATCC, TIB-161).

Techniques: Expressing, Derivative Assay

Journal: Cell Cycle

Article Title: Exogenous expression of SAMHD1 inhibits proliferation and induces apoptosis in cutaneous T-cell lymphoma-derived HuT78 cells

doi: 10.1080/15384101.2016.1261226

Figure Lengend Snippet: Exogenous SAMHD1 expression inhibits HuT78 cell growth and proliferation. HuT78 cells expressing hemagglutinin (HA)-tagged SAMHD1 were generated via lentiviral vector-mediated transduction. (A) Exogenous expression of SAMHD1 was validated by immunoblotting analysis using antibodies to HA (SAMHD1) and GAPDH (loading control). (B) Total RNA was isolated from HuT78 vector or SAMHD1-expressing cells and qPCR analysis was performed to quantify the relative SAMHD1 mRNA levels. GAPDH mRNA levels were quantified as internal control. (C) HuT78 vector control and SAMHD1 expressing cells were seeded in triplicate in 24-well plates at a density of 1 × 104 cells/well in 1 ml culture media on day 0 and live cells were counted on indicated days via trypan-blue exclusion method. (D) Cell lines were seeded on day 0 in 4 replicates in 96-well plates at density of 1 × 103 cells/well in 100 µl and cell proliferation was determined on indicated days utilizing an MTS-based proliferation assay. All the data presented are representative of 3 or more independent experiments. (C-D) ***, p < 0.0001.

Article Snippet: Generation of HuT78 cell lines with stable expression of SAMHD1 Human CD4 + T-lymphocytic cell line HuT78 derived from an Sézary syndrome patient was obtained from the American Type Culture Collection (ATCC, TIB-161).

Techniques: Expressing, Generated, Plasmid Preparation, Transduction, Western Blot, Control, Isolation, Proliferation Assay

Journal: Cell Cycle

Article Title: Exogenous expression of SAMHD1 inhibits proliferation and induces apoptosis in cutaneous T-cell lymphoma-derived HuT78 cells

doi: 10.1080/15384101.2016.1261226

Figure Lengend Snippet: Exogenous SAMHD1 expression in HuT78 cells results in reduced colony formation. HuT78 vector control, and SAMHD1-expressing cells were plated in duplicate in 6-well plates at a density of 1 × 103 cells/well in 1 ml methyl cellulose medium and were left in culture for 10 d. (A) Numbers of colonies were counted after 10 d following standard counting criteria (all colonies with >25 cells were counted). Data is presented as % of colony yield relative to vector control cells (left panel). **, p < 0.005, Representative images (20 × magnification) of colonies formed after 10 d (right panel). (B) Numbers of colonies of different sizes were counted after 10 d based on following standard counting criteria (small: <25 cells; medium: 25–50 cells, and large: >50 cells). Data is presented as percentages of colony yield relative to vector control cells. All the data presented are representative of 3 independent experiments.

Article Snippet: Generation of HuT78 cell lines with stable expression of SAMHD1 Human CD4 + T-lymphocytic cell line HuT78 derived from an Sézary syndrome patient was obtained from the American Type Culture Collection (ATCC, TIB-161).

Techniques: Expressing, Plasmid Preparation, Control

Journal: Cell Cycle

Article Title: Exogenous expression of SAMHD1 inhibits proliferation and induces apoptosis in cutaneous T-cell lymphoma-derived HuT78 cells

doi: 10.1080/15384101.2016.1261226

Figure Lengend Snippet: Exogenous SAMHD1 expression induces apoptosis and caspase-3/7 activity via activation of extrinsic apoptosis signaling. HuT78 vector control and SAMHD1 expressing cells were seeded at density of 2 × 104 cells per 6-cm dish containing 5 ml growth media on day 0 following trypan-blue exclusion method. (A) Seven days post-seeding, cells in triplicate, were stained with propidium iodide (PI) and cell cycle analysis was performed via flow cytometry. Percentages of cells in G1/G0, S, and G2/M phases of cell cycle are presented. (B) Percentage of cells in Sub-G1 phase are presented. (C) HuT78 vector control and SAMHD1-expressing cells were seeded at density of 2 × 104 cells per 6-cm dish containing 5 ml growth media on day 0 following trypan-blue exclusion method. Seven days post-seeding, cells were stained with annexin V-PE and 7-AAD in triplicate and analyzed by flow cytometry. Representative flow cytometric profiles are presented (left panel). The percentages of non-apoptotic cells, early apoptotic cells, and late apoptotic cells were quantified as presented (right panel). (D) On day 7 post-seeding, cell lysates from all cell lines were collected and western analysis was performed using the antibodies to caspase-8, caspase-9, caspase-3, PARP, HA (SAMHD1), and GAPDH (loading control). (E) On day 7 post-seeding, 1 × 104 cells per cell line were collected in 4 replicates and incubated in 100 µl of Caspase-Glo 3/7 reagent at room temperature in dark for 1 hour. Caspase-3/7 activity was then determined by measuring luminescence values. All the data presented are representative of 3 independent experiments. B, C, E, ***, p < 0.001.

Article Snippet: Generation of HuT78 cell lines with stable expression of SAMHD1 Human CD4 + T-lymphocytic cell line HuT78 derived from an Sézary syndrome patient was obtained from the American Type Culture Collection (ATCC, TIB-161).

Techniques: Expressing, Activity Assay, Activation Assay, Plasmid Preparation, Control, Staining, Cell Cycle Assay, Flow Cytometry, Western Blot, Incubation

Journal: Cell Cycle

Article Title: Exogenous expression of SAMHD1 inhibits proliferation and induces apoptosis in cutaneous T-cell lymphoma-derived HuT78 cells

doi: 10.1080/15384101.2016.1261226

Figure Lengend Snippet: Exogenous SAMHD1 expression in HuT78 cells significantly increases Fas-L induced apoptosis and caspase-3/7 activity. (A) HuT78 vector control and SAMHD1-expressing cells were treated with 100 ng/ml Fas-L for 48 hours. After 48 hours of treatment, all the cells in triplicate were stained with annexin V-PE and 7-AminoactinomycinD (7-AAD) followed by flow cytometry. Representative flow cytometric profiles are presented (left panel). The percentage of non-apoptotic cells, early apoptotic cells, and late apoptotic cells were quantified as presented (right panel). (B) HuT78 vector control and SAMHD1-expressing cells were treated with 100 ng/ml Fas-L for 48 hours. Post-treatment, 1 × 104 cells per cell line were collected in triplicate and incubated in 100 µl of Caspase-Glo 3/7 reagent at room temperature in dark for 1 hour. Caspase-3/7 activity was then determined by measuring luminescence values. C, HuT78 vector control and SAMHD1-expressing cells were treated with Fas-L (100 ng/ml) for 48 hours. Post-treatment, cell lysates from all cell lines were collected and immunoblotting was performed using the antibodies to caspase-8, caspase-3, PARP, and GAPDH (loading control). All the data presented are representative of 3 independent experiments. A–B, ***, p < 0.001.

Article Snippet: Generation of HuT78 cell lines with stable expression of SAMHD1 Human CD4 + T-lymphocytic cell line HuT78 derived from an Sézary syndrome patient was obtained from the American Type Culture Collection (ATCC, TIB-161).

Techniques: Expressing, Activity Assay, Plasmid Preparation, Control, Staining, Flow Cytometry, Incubation, Western Blot

Journal: Cell Cycle

Article Title: Exogenous expression of SAMHD1 inhibits proliferation and induces apoptosis in cutaneous T-cell lymphoma-derived HuT78 cells

doi: 10.1080/15384101.2016.1261226

Figure Lengend Snippet: Exogenous SAMHD1 expression in HuT78 cells reduces the levels of cFLIPS protein and mRNA. HuT78 vector control and SAMHD1-expressing cells were seeded at density of 2 × 104 cells per 6-cm dish containing 5 ml growth media on day 0 following trypan-blue exclusion method. (A) On day 7 post-seeding, cell lysates were collected and immunoblotting analysis was performed for cFLIP, HA (SAMHD1) and GAPDH (loading control) (left panel). Densitometry was performed using ImageJ to quantify the relative cFLIPL and cFLIPS protein levels (right panel). ***, p < 0.001 (B) On day 7 post-seeding, total RNA was isolated from HuT78 vector or SAMHD1-expressing cells and qPCR analysis was performed to quantify the relative cFLIPL and cFLIPS mRNA levels. GAPDH mRNA levels were quantified as internal control. All the data presented are representative of 3 independent experiments. *, p < 0.05.

Article Snippet: Generation of HuT78 cell lines with stable expression of SAMHD1 Human CD4 + T-lymphocytic cell line HuT78 derived from an Sézary syndrome patient was obtained from the American Type Culture Collection (ATCC, TIB-161).

Techniques: Expressing, Plasmid Preparation, Control, Western Blot, Isolation

Journal: Cell Cycle

Article Title: Exogenous expression of SAMHD1 inhibits proliferation and induces apoptosis in cutaneous T-cell lymphoma-derived HuT78 cells

doi: 10.1080/15384101.2016.1261226

Figure Lengend Snippet: Summary of the findings and proposed mechanisms. Our data indicate that increased SAMHD1 expression inhibits growth and proliferation in CTCL-derived CD4+ T-cells by inducing spontaneous and Fas-L stimulated apoptosis. SAMHD1 expression also diminishes colony forming potential in these cells. Furthermore, increased SAMHD1 expression also significantly reduces the mRNA and protein levels of cFLIPS, a key anti-apoptotic protein. Complete mechanisms by which SAMHD1 regulates cFLIPS expression, apoptosis and colony formation in HuT78 cells remain to be understood.

Article Snippet: Generation of HuT78 cell lines with stable expression of SAMHD1 Human CD4 + T-lymphocytic cell line HuT78 derived from an Sézary syndrome patient was obtained from the American Type Culture Collection (ATCC, TIB-161).

Techniques: Expressing, Derivative Assay