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ZeptoMetrix corporation htlv 1 p19 gag production

Cellular m 6 A depletion decreases sense-derived viral genes and increases hbz expression. C91/PL (HTLV-1-transformed T-cell line) cells were treated with vehicle or titrating amounts (20 µM, 40 µM, 60 µM, and 80 µM) of STM2457 for 72 h. ( A ) The level of m 6 A-modified mRNA in the cell was quantified using an ELISA. ( B ) Western blot analysis was used to measure viral protein (Env, Tax, <t>p19</t> [Gag], and Hbz) expression. β-actin was used as a loading control. Relative band intensity was quantified using ImageJ. Protein quantification relative to DMSO (normalized to β-actin) is depicted below each western blot. ( C ) Virus release in the cell supernatant was measured using a p19 Gag ELISA. ( D ) Infectious virus release was quantified by measuring TdTomato expression in JET cells through flow cytometry. ( E ) RNA was isolated, and qRT-PCR was used to measure sense-derived transcripts ( tax, gag, and env ) and antisense-derived ( hbz ) transcripts. HTLV-1-transformed T-cell lines (SLB-1, Hut-102) were treated with or without 60 µM STM2457 for 72 h. ( F ) RNA was isolated and qRT-PCR was used to measure sense-derived transcripts ( tax, gag, and env ) and antisense-derived ( hbz ) transcripts. ( G ) Western blot analysis was used to measure viral protein (Env, Tax, p19 [Gag], and Hbz) expression. β-actin was used as a loading control. Relative band intensity was quantified using ImageJ. Protein quantification relative to DMSO (normalized to β-actin) is depicted below each western blot. ( H ) Virus release in the cell supernatant was measured using a p19 Gag ELISA. Statistical significance was determined using a Student’s t -test; * P ≤ 0.05, ** P ≤ 0.01, *** P ≤ 0.001, **** P ≤ 0.0001.

Htlv 1 P19 Gag Production, supplied by ZeptoMetrix corporation, used in various techniques. Bioz Stars score: 94/100, based on 43 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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htlv 1 p19 gag production - by Bioz Stars, 2026-06

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1) Product Images from "YTHDF1 and YTHDC1 m 6 A reader proteins regulate HTLV-1 tax and hbz activity"

Article Title: YTHDF1 and YTHDC1 m 6 A reader proteins regulate HTLV-1 tax and hbz activity

Journal: Journal of Virology

doi: 10.1128/jvi.02063-24

Figure Legend Snippet: Cellular m 6 A depletion decreases sense-derived viral genes and increases hbz expression. C91/PL (HTLV-1-transformed T-cell line) cells were treated with vehicle or titrating amounts (20 µM, 40 µM, 60 µM, and 80 µM) of STM2457 for 72 h. ( A ) The level of m 6 A-modified mRNA in the cell was quantified using an ELISA. ( B ) Western blot analysis was used to measure viral protein (Env, Tax, p19 [Gag], and Hbz) expression. β-actin was used as a loading control. Relative band intensity was quantified using ImageJ. Protein quantification relative to DMSO (normalized to β-actin) is depicted below each western blot. ( C ) Virus release in the cell supernatant was measured using a p19 Gag ELISA. ( D ) Infectious virus release was quantified by measuring TdTomato expression in JET cells through flow cytometry. ( E ) RNA was isolated, and qRT-PCR was used to measure sense-derived transcripts ( tax, gag, and env ) and antisense-derived ( hbz ) transcripts. HTLV-1-transformed T-cell lines (SLB-1, Hut-102) were treated with or without 60 µM STM2457 for 72 h. ( F ) RNA was isolated and qRT-PCR was used to measure sense-derived transcripts ( tax, gag, and env ) and antisense-derived ( hbz ) transcripts. ( G ) Western blot analysis was used to measure viral protein (Env, Tax, p19 [Gag], and Hbz) expression. β-actin was used as a loading control. Relative band intensity was quantified using ImageJ. Protein quantification relative to DMSO (normalized to β-actin) is depicted below each western blot. ( H ) Virus release in the cell supernatant was measured using a p19 Gag ELISA. Statistical significance was determined using a Student’s t -test; * P ≤ 0.05, ** P ≤ 0.01, *** P ≤ 0.001, **** P ≤ 0.0001.

Techniques Used: Derivative Assay, Expressing, Transformation Assay, Modification, Enzyme-linked Immunosorbent Assay, Western Blot, Control, Virus, Flow Cytometry, Isolation, Quantitative RT-PCR

YTHDF1 decreases sense-derived and increases antisense-derived viral transcripts. HEK293T cells were treated with or without 60 µM STM2457 for 72 h and then transfected with LTR-1-luc, rtk-luc, HTLV-1 proviral DNA, and ±100 ng FLAG-YTHDF1 expression vector. Cells were collected 72 h post-transfection. ( A ) Cells were lysed in passive lysis buffer and firefly luciferase activity was quantified relative to renilla luciferase. ( B ) RNA was isolated, and qRT-PCR was used to measure sense-derived transcripts ( tax, gag, and env ) and antisense-derived ( hbz ) transcripts. ( C ) Western blot analysis was used to measure viral protein (Env, Tax, p19 [Gag], and Hbz) expression. β-actin was used as a loading control. Relative band intensity was quantified using ImageJ. Protein quantification relative to no YTHDF1 (lane 1; normalized to β-actin) is depicted below each western blot. ( D ) Virus release in the cell supernatant was measured using a p19 Gag ELISA. ( E ) Western blot analysis was used to measure transcription factors SP1, JunD, MEF-2A, and MEF-2C. Antibodies against YTHDF1 and β-actin (loading control) were also used. The YTHDF1 and β-actin blots in panel E are duplicates of those depicted in panel C as they are from the same experiment. Relative band intensity was quantified using ImageJ. Protein quantification relative to no YTHDF1 (lane 1; normalized to β-actin) is depicted below each western blot. Statistical significance was determined using a Student’s t -test; * P ≤ 0.05, ** P ≤ 0.01.

Figure Legend Snippet: YTHDF1 decreases sense-derived and increases antisense-derived viral transcripts. HEK293T cells were treated with or without 60 µM STM2457 for 72 h and then transfected with LTR-1-luc, rtk-luc, HTLV-1 proviral DNA, and ±100 ng FLAG-YTHDF1 expression vector. Cells were collected 72 h post-transfection. ( A ) Cells were lysed in passive lysis buffer and firefly luciferase activity was quantified relative to renilla luciferase. ( B ) RNA was isolated, and qRT-PCR was used to measure sense-derived transcripts ( tax, gag, and env ) and antisense-derived ( hbz ) transcripts. ( C ) Western blot analysis was used to measure viral protein (Env, Tax, p19 [Gag], and Hbz) expression. β-actin was used as a loading control. Relative band intensity was quantified using ImageJ. Protein quantification relative to no YTHDF1 (lane 1; normalized to β-actin) is depicted below each western blot. ( D ) Virus release in the cell supernatant was measured using a p19 Gag ELISA. ( E ) Western blot analysis was used to measure transcription factors SP1, JunD, MEF-2A, and MEF-2C. Antibodies against YTHDF1 and β-actin (loading control) were also used. The YTHDF1 and β-actin blots in panel E are duplicates of those depicted in panel C as they are from the same experiment. Relative band intensity was quantified using ImageJ. Protein quantification relative to no YTHDF1 (lane 1; normalized to β-actin) is depicted below each western blot. Statistical significance was determined using a Student’s t -test; * P ≤ 0.05, ** P ≤ 0.01.

Techniques Used: Derivative Assay, Transfection, Expressing, Plasmid Preparation, Lysis, Luciferase, Activity Assay, Isolation, Quantitative RT-PCR, Western Blot, Control, Virus, Enzyme-linked Immunosorbent Assay

Knockdown of YTHDF1 increases sense-derived and decreases antisense-derived viral transcripts. SLB-1 and ATL-ED cells were treated with or without 60 µM STM2457 for 72 h. Cells were then transduced with shControl and shYTHDF1 lentivirus. ( A and D ) Western blot analysis was used to measure viral protein (Env, Tax, p19 [Gag], and Hbz), YTHDF1 and β-actin (loading control). Relative band intensity was quantified using ImageJ. Protein quantification relative to DMSO, shControl (lane 1; normalized to β-actin) is depicted below each western blot. ( B ) Virus release in the cell supernatant (of SLB-1 cells) was measured using a p19 Gag ELISA. ( C and E ) RNA was isolated and qRT-PCR was used to measure the transcript levels of tax , env , gag , and hbz relative to gapdh . ( F ) SLB-1 cells and ( G ) ATL-ED cells were subjected to immunoblot to measure YTHDF1, JunD, SP1, MEF-2A, MEF-2C, and β-actin (loading control). The YTHDF1 and β-actin blots in panels F and G are duplicates of those depicted in panels A and D, respectively, as they are from the same experiment. Relative band intensity was quantified using ImageJ. Protein quantification relative to shControl, DMSO (lane 1; normalized to β-actin) is depicted below each western blot. Statistical significance was determined using a Student’s t -test; * P ≤ 0.05, ** P ≤ 0.01.

Figure Legend Snippet: Knockdown of YTHDF1 increases sense-derived and decreases antisense-derived viral transcripts. SLB-1 and ATL-ED cells were treated with or without 60 µM STM2457 for 72 h. Cells were then transduced with shControl and shYTHDF1 lentivirus. ( A and D ) Western blot analysis was used to measure viral protein (Env, Tax, p19 [Gag], and Hbz), YTHDF1 and β-actin (loading control). Relative band intensity was quantified using ImageJ. Protein quantification relative to DMSO, shControl (lane 1; normalized to β-actin) is depicted below each western blot. ( B ) Virus release in the cell supernatant (of SLB-1 cells) was measured using a p19 Gag ELISA. ( C and E ) RNA was isolated and qRT-PCR was used to measure the transcript levels of tax , env , gag , and hbz relative to gapdh . ( F ) SLB-1 cells and ( G ) ATL-ED cells were subjected to immunoblot to measure YTHDF1, JunD, SP1, MEF-2A, MEF-2C, and β-actin (loading control). The YTHDF1 and β-actin blots in panels F and G are duplicates of those depicted in panels A and D, respectively, as they are from the same experiment. Relative band intensity was quantified using ImageJ. Protein quantification relative to shControl, DMSO (lane 1; normalized to β-actin) is depicted below each western blot. Statistical significance was determined using a Student’s t -test; * P ≤ 0.05, ** P ≤ 0.01.

Techniques Used: Knockdown, Derivative Assay, Transduction, Western Blot, Control, Virus, Enzyme-linked Immunosorbent Assay, Isolation, Quantitative RT-PCR

Loss of YTHDC1 decreases viral transcripts. SLB-1 and ATL-ED cells were treated with or without 60 µM STM2457 for 72 h and transduced with shControl and shYTHDF1 lentivirus. ( A ) SLB-1 cells were subjected to western blot analysis to measure YTHDF1, Env, Tax, p19 (Gag), Hbz, and β-actin (loading control). Relative band intensity was quantified using ImageJ. Protein quantification relative to shControl, DMSO (normalized to β-actin) is depicted below each western blot. ( B ) Virus release in the cell supernatant was measured using a p19 Gag ELISA. ( C ) RNA was isolated and qRT-PCR was used to measure transcript levels of tax , gag , env, and hbz relative to gapdh . ( D ) ATL-ED cells were subjected to western blot analysis to measure YTHDF1, Hbz, and β-actin (loading control). Relative band intensity was quantified using ImageJ. Protein quantification relative to shControl; DMSO (normalized to β-actin) is depicted below each western blot. ( E ) RNA was isolated, and qRT-PCR was used to measure transcript levels of hbz relative to gapdh . Statistical significance was determined using a Student’s t -test; * P ≤ 0.05, ** P ≤ 0.01.

Figure Legend Snippet: Loss of YTHDC1 decreases viral transcripts. SLB-1 and ATL-ED cells were treated with or without 60 µM STM2457 for 72 h and transduced with shControl and shYTHDF1 lentivirus. ( A ) SLB-1 cells were subjected to western blot analysis to measure YTHDF1, Env, Tax, p19 (Gag), Hbz, and β-actin (loading control). Relative band intensity was quantified using ImageJ. Protein quantification relative to shControl, DMSO (normalized to β-actin) is depicted below each western blot. ( B ) Virus release in the cell supernatant was measured using a p19 Gag ELISA. ( C ) RNA was isolated and qRT-PCR was used to measure transcript levels of tax , gag , env, and hbz relative to gapdh . ( D ) ATL-ED cells were subjected to western blot analysis to measure YTHDF1, Hbz, and β-actin (loading control). Relative band intensity was quantified using ImageJ. Protein quantification relative to shControl; DMSO (normalized to β-actin) is depicted below each western blot. ( E ) RNA was isolated, and qRT-PCR was used to measure transcript levels of hbz relative to gapdh . Statistical significance was determined using a Student’s t -test; * P ≤ 0.05, ** P ≤ 0.01.

Techniques Used: Transduction, Western Blot, Control, Virus, Enzyme-linked Immunosorbent Assay, Isolation, Quantitative RT-PCR

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Journal: Journal of Virology

Article Title: YTHDF1 and YTHDC1 m 6 A reader proteins regulate HTLV-1 tax and hbz activity

doi: 10.1128/jvi.02063-24

Figure Lengend Snippet: Cellular m 6 A depletion decreases sense-derived viral genes and increases hbz expression. C91/PL (HTLV-1-transformed T-cell line) cells were treated with vehicle or titrating amounts (20 µM, 40 µM, 60 µM, and 80 µM) of STM2457 for 72 h. ( A ) The level of m 6 A-modified mRNA in the cell was quantified using an ELISA. ( B ) Western blot analysis was used to measure viral protein (Env, Tax, p19 [Gag], and Hbz) expression. β-actin was used as a loading control. Relative band intensity was quantified using ImageJ. Protein quantification relative to DMSO (normalized to β-actin) is depicted below each western blot. ( C ) Virus release in the cell supernatant was measured using a p19 Gag ELISA. ( D ) Infectious virus release was quantified by measuring TdTomato expression in JET cells through flow cytometry. ( E ) RNA was isolated, and qRT-PCR was used to measure sense-derived transcripts ( tax, gag, and env ) and antisense-derived ( hbz ) transcripts. HTLV-1-transformed T-cell lines (SLB-1, Hut-102) were treated with or without 60 µM STM2457 for 72 h. ( F ) RNA was isolated and qRT-PCR was used to measure sense-derived transcripts ( tax, gag, and env ) and antisense-derived ( hbz ) transcripts. ( G ) Western blot analysis was used to measure viral protein (Env, Tax, p19 [Gag], and Hbz) expression. β-actin was used as a loading control. Relative band intensity was quantified using ImageJ. Protein quantification relative to DMSO (normalized to β-actin) is depicted below each western blot. ( H ) Virus release in the cell supernatant was measured using a p19 Gag ELISA. Statistical significance was determined using a Student’s t -test; * P ≤ 0.05, ** P ≤ 0.01, *** P ≤ 0.001, **** P ≤ 0.0001.

Article Snippet: Twenty-four hours later, the supernatant was collected to measure HTLV-1 p19 Gag production using the RETROTEK HTLV p19 Antigen ELISA (ZeptoMetrix Corporation, Buffalo, NY) according to the manufacturer’s instructions.

Techniques: Derivative Assay, Expressing, Transformation Assay, Modification, Enzyme-linked Immunosorbent Assay, Western Blot, Control, Virus, Flow Cytometry, Isolation, Quantitative RT-PCR

Journal: Journal of Virology

Article Title: YTHDF1 and YTHDC1 m 6 A reader proteins regulate HTLV-1 tax and hbz activity

doi: 10.1128/jvi.02063-24

Figure Lengend Snippet: YTHDF1 decreases sense-derived and increases antisense-derived viral transcripts. HEK293T cells were treated with or without 60 µM STM2457 for 72 h and then transfected with LTR-1-luc, rtk-luc, HTLV-1 proviral DNA, and ±100 ng FLAG-YTHDF1 expression vector. Cells were collected 72 h post-transfection. ( A ) Cells were lysed in passive lysis buffer and firefly luciferase activity was quantified relative to renilla luciferase. ( B ) RNA was isolated, and qRT-PCR was used to measure sense-derived transcripts ( tax, gag, and env ) and antisense-derived ( hbz ) transcripts. ( C ) Western blot analysis was used to measure viral protein (Env, Tax, p19 [Gag], and Hbz) expression. β-actin was used as a loading control. Relative band intensity was quantified using ImageJ. Protein quantification relative to no YTHDF1 (lane 1; normalized to β-actin) is depicted below each western blot. ( D ) Virus release in the cell supernatant was measured using a p19 Gag ELISA. ( E ) Western blot analysis was used to measure transcription factors SP1, JunD, MEF-2A, and MEF-2C. Antibodies against YTHDF1 and β-actin (loading control) were also used. The YTHDF1 and β-actin blots in panel E are duplicates of those depicted in panel C as they are from the same experiment. Relative band intensity was quantified using ImageJ. Protein quantification relative to no YTHDF1 (lane 1; normalized to β-actin) is depicted below each western blot. Statistical significance was determined using a Student’s t -test; * P ≤ 0.05, ** P ≤ 0.01.

Techniques: Derivative Assay, Transfection, Expressing, Plasmid Preparation, Lysis, Luciferase, Activity Assay, Isolation, Quantitative RT-PCR, Western Blot, Control, Virus, Enzyme-linked Immunosorbent Assay

Journal: Journal of Virology

Article Title: YTHDF1 and YTHDC1 m 6 A reader proteins regulate HTLV-1 tax and hbz activity

doi: 10.1128/jvi.02063-24

Figure Lengend Snippet: Knockdown of YTHDF1 increases sense-derived and decreases antisense-derived viral transcripts. SLB-1 and ATL-ED cells were treated with or without 60 µM STM2457 for 72 h. Cells were then transduced with shControl and shYTHDF1 lentivirus. ( A and D ) Western blot analysis was used to measure viral protein (Env, Tax, p19 [Gag], and Hbz), YTHDF1 and β-actin (loading control). Relative band intensity was quantified using ImageJ. Protein quantification relative to DMSO, shControl (lane 1; normalized to β-actin) is depicted below each western blot. ( B ) Virus release in the cell supernatant (of SLB-1 cells) was measured using a p19 Gag ELISA. ( C and E ) RNA was isolated and qRT-PCR was used to measure the transcript levels of tax , env , gag , and hbz relative to gapdh . ( F ) SLB-1 cells and ( G ) ATL-ED cells were subjected to immunoblot to measure YTHDF1, JunD, SP1, MEF-2A, MEF-2C, and β-actin (loading control). The YTHDF1 and β-actin blots in panels F and G are duplicates of those depicted in panels A and D, respectively, as they are from the same experiment. Relative band intensity was quantified using ImageJ. Protein quantification relative to shControl, DMSO (lane 1; normalized to β-actin) is depicted below each western blot. Statistical significance was determined using a Student’s t -test; * P ≤ 0.05, ** P ≤ 0.01.

Techniques: Knockdown, Derivative Assay, Transduction, Western Blot, Control, Virus, Enzyme-linked Immunosorbent Assay, Isolation, Quantitative RT-PCR

Journal: Journal of Virology

Article Title: YTHDF1 and YTHDC1 m 6 A reader proteins regulate HTLV-1 tax and hbz activity

doi: 10.1128/jvi.02063-24

Figure Lengend Snippet: Loss of YTHDC1 decreases viral transcripts. SLB-1 and ATL-ED cells were treated with or without 60 µM STM2457 for 72 h and transduced with shControl and shYTHDF1 lentivirus. ( A ) SLB-1 cells were subjected to western blot analysis to measure YTHDF1, Env, Tax, p19 (Gag), Hbz, and β-actin (loading control). Relative band intensity was quantified using ImageJ. Protein quantification relative to shControl, DMSO (normalized to β-actin) is depicted below each western blot. ( B ) Virus release in the cell supernatant was measured using a p19 Gag ELISA. ( C ) RNA was isolated and qRT-PCR was used to measure transcript levels of tax , gag , env, and hbz relative to gapdh . ( D ) ATL-ED cells were subjected to western blot analysis to measure YTHDF1, Hbz, and β-actin (loading control). Relative band intensity was quantified using ImageJ. Protein quantification relative to shControl; DMSO (normalized to β-actin) is depicted below each western blot. ( E ) RNA was isolated, and qRT-PCR was used to measure transcript levels of hbz relative to gapdh . Statistical significance was determined using a Student’s t -test; * P ≤ 0.05, ** P ≤ 0.01.

Techniques: Transduction, Western Blot, Control, Virus, Enzyme-linked Immunosorbent Assay, Isolation, Quantitative RT-PCR

Hbz mutant constructs were generated in the context of the HTLV-1 proviral plasmid ACHneo. (A) HEK293T cells were co-transfected with pcDNA3.1(+) empty vector, WT, M3, ΔHbz, M3.ΔHbz, or SAm proviral plasmid and an HTLV-1 LTR-firefly luciferase construct. 48h post-transfection, cells and supernatant were collected for luciferase assay and ELISA to detect HTLV-1 p19 Gag (B), respectively. (C) RNA was extracted from transfected cells for cDNA synthesis and qPCR to detect hbz mRNA levels. Hbz copy number is shown normalized to 1 x 10 6 gapdh copies. Graphs represent data generated from duplicate samples and error bars represent standard deviation (SD). Data are representative of at least three experimental repeats.

Journal: PLOS Pathogens

Article Title: HTLV-1 Hbz protein, but not hbz mRNA secondary structure, is critical for viral persistence and disease development

doi: 10.1371/journal.ppat.1011459

Figure Lengend Snippet: Hbz mutant constructs were generated in the context of the HTLV-1 proviral plasmid ACHneo. (A) HEK293T cells were co-transfected with pcDNA3.1(+) empty vector, WT, M3, ΔHbz, M3.ΔHbz, or SAm proviral plasmid and an HTLV-1 LTR-firefly luciferase construct. 48h post-transfection, cells and supernatant were collected for luciferase assay and ELISA to detect HTLV-1 p19 Gag (B), respectively. (C) RNA was extracted from transfected cells for cDNA synthesis and qPCR to detect hbz mRNA levels. Hbz copy number is shown normalized to 1 x 10 6 gapdh copies. Graphs represent data generated from duplicate samples and error bars represent standard deviation (SD). Data are representative of at least three experimental repeats.

Article Snippet: Approximately two weeks after G418 selection, cell supernatant was collected to measure HTLV-1 p19 Gag production by ELISA (ZeptoMetrix Corporation, Buffalo, NY).

Techniques: Mutagenesis, Construct, Generated, Plasmid Preparation, Transfection, Luciferase, Enzyme-linked Immunosorbent Assay, cDNA Synthesis, Standard Deviation

WT, M3, ΔHbz, M3.ΔHbz, and SAm producer cells were generated and (A) culture supernatant was collected for p19 Gag ELISA. Statistical significance was determined by one-way ANOVA with Dunnett’s multiple comparisons test. *P ≤ 0.05, ***P ≤ 0.001, ****P ≤ 0.0001. (B) RNA was extracted from 729.B parental, WT, and mutant producer clones for cDNA synthesis and qPCR to detect hbz mRNA expression. Hbz copy number is shown normalized to 1 x 10 6 gapdh copies. (C) Total protein was quantified in cell lysates from parental, WT, and mutant 729 cell lines, and equal amounts were loaded onto an SDS-PAGE gel for immunoblotting analysis. β-actin is shown as a loading control, and arrows are used to distinguish background bands from those corresponding to Hbz protein. Graphs represent data generated from duplicate samples and error bars represent SD. Data are representative of at least three experimental repeats.

Journal: PLOS Pathogens

Article Title: HTLV-1 Hbz protein, but not hbz mRNA secondary structure, is critical for viral persistence and disease development

doi: 10.1371/journal.ppat.1011459

Figure Lengend Snippet: WT, M3, ΔHbz, M3.ΔHbz, and SAm producer cells were generated and (A) culture supernatant was collected for p19 Gag ELISA. Statistical significance was determined by one-way ANOVA with Dunnett’s multiple comparisons test. *P ≤ 0.05, ***P ≤ 0.001, ****P ≤ 0.0001. (B) RNA was extracted from 729.B parental, WT, and mutant producer clones for cDNA synthesis and qPCR to detect hbz mRNA expression. Hbz copy number is shown normalized to 1 x 10 6 gapdh copies. (C) Total protein was quantified in cell lysates from parental, WT, and mutant 729 cell lines, and equal amounts were loaded onto an SDS-PAGE gel for immunoblotting analysis. β-actin is shown as a loading control, and arrows are used to distinguish background bands from those corresponding to Hbz protein. Graphs represent data generated from duplicate samples and error bars represent SD. Data are representative of at least three experimental repeats.

Article Snippet: Approximately two weeks after G418 selection, cell supernatant was collected to measure HTLV-1 p19 Gag production by ELISA (ZeptoMetrix Corporation, Buffalo, NY).

Techniques: Generated, Enzyme-linked Immunosorbent Assay, Mutagenesis, Clone Assay, cDNA Synthesis, Expressing, SDS Page, Western Blot, Control

$1 x 10 6 lethally irradiated 729.WT, 729.M3, 729.ΔHbz, 729.M3.ΔHbz, or 729.SAm producer cells were co-cultured in 24-well plates with 2 x 10 6 freshly isolated hPBMCs from healthy donors in two independent experiments. Long-term immortalization results and characterization of newly immortalized PBL cell lines are shown from a representative experiment. (A) Cells in the co-culture were provided 10 U/mL hIL-2 once per week with fresh media. T-cell immortalization was determined with weekly viable cell counts by trypan blue exclusion. (B) Cell supernatant was collected for p19 Gag ELISA at weekly intervals, beginning at Week 4, to determine virion production. Each time point depicts data collected from three random, independent wells (technical replicates) and error bars represent SD. RNA was extracted from PBL cell lines immortalized by WT, M3, ΔHbz, M3.ΔHbz, and SAm viruses for cDNA synthesis and qPCR to detect (C) spliced hbz mRNA expression and (D) unspliced hbz RNA expression. Hbz copy number is shown normalized to 1 x 10 6 gapdh copies. Unspliced hbz is shown as fold change relative to WT. Data were generated from duplicate samples and error bars represent SD. The number of newly immortalized cell lines that were analyzed for each virus are as follows: WT n = 4, M3 n = 2, ΔHbz n = 2, M3.ΔHbz n = 3, and SAm n = 3. (E) Total protein was quantified in cell lysates from WT and mutant cell lines and equal amounts were loaded onto an SDS-PAGE gel for immunoblotting analysis. β-actin is shown as a loading control, and arrows are used to distinguish background bands from those corresponding to Hbz protein. (F) Phenotyping of immortalized T-cells was performed by flow cytometry using antibodies against CD3, CD4, and CD8 surface markers. The average percentage of CD4 and CD8 T-cells in each mutant condition is depicted below. (G) Cell lines were collected for nuclear/cytoplasmic cell fractionation followed by RNA extraction, cDNA synthesis, and qPCR to detect spliced hbz in each fraction. Each dot represents an individual cell line, generated from duplicate samples and bars represent the mean. An ATL-derived cell line (ATL-ED) was used as a positive control. (H) Proliferation was measured by MTS assay. Error bars represent the SD of three technical replicates. Data in all panels are representative of at least three experimental repeats.$

Journal: PLOS Pathogens

Article Title: HTLV-1 Hbz protein, but not hbz mRNA secondary structure, is critical for viral persistence and disease development

doi: 10.1371/journal.ppat.1011459

Figure Lengend Snippet: 1 x 10 6 lethally irradiated 729.WT, 729.M3, 729.ΔHbz, 729.M3.ΔHbz, or 729.SAm producer cells were co-cultured in 24-well plates with 2 x 10 6 freshly isolated hPBMCs from healthy donors in two independent experiments. Long-term immortalization results and characterization of newly immortalized PBL cell lines are shown from a representative experiment. (A) Cells in the co-culture were provided 10 U/mL hIL-2 once per week with fresh media. T-cell immortalization was determined with weekly viable cell counts by trypan blue exclusion. (B) Cell supernatant was collected for p19 Gag ELISA at weekly intervals, beginning at Week 4, to determine virion production. Each time point depicts data collected from three random, independent wells (technical replicates) and error bars represent SD. RNA was extracted from PBL cell lines immortalized by WT, M3, ΔHbz, M3.ΔHbz, and SAm viruses for cDNA synthesis and qPCR to detect (C) spliced hbz mRNA expression and (D) unspliced hbz RNA expression. Hbz copy number is shown normalized to 1 x 10 6 gapdh copies. Unspliced hbz is shown as fold change relative to WT. Data were generated from duplicate samples and error bars represent SD. The number of newly immortalized cell lines that were analyzed for each virus are as follows: WT n = 4, M3 n = 2, ΔHbz n = 2, M3.ΔHbz n = 3, and SAm n = 3. (E) Total protein was quantified in cell lysates from WT and mutant cell lines and equal amounts were loaded onto an SDS-PAGE gel for immunoblotting analysis. β-actin is shown as a loading control, and arrows are used to distinguish background bands from those corresponding to Hbz protein. (F) Phenotyping of immortalized T-cells was performed by flow cytometry using antibodies against CD3, CD4, and CD8 surface markers. The average percentage of CD4 and CD8 T-cells in each mutant condition is depicted below. (G) Cell lines were collected for nuclear/cytoplasmic cell fractionation followed by RNA extraction, cDNA synthesis, and qPCR to detect spliced hbz in each fraction. Each dot represents an individual cell line, generated from duplicate samples and bars represent the mean. An ATL-derived cell line (ATL-ED) was used as a positive control. (H) Proliferation was measured by MTS assay. Error bars represent the SD of three technical replicates. Data in all panels are representative of at least three experimental repeats.

Article Snippet: Approximately two weeks after G418 selection, cell supernatant was collected to measure HTLV-1 p19 Gag production by ELISA (ZeptoMetrix Corporation, Buffalo, NY).

Techniques: Irradiation, Cell Culture, Isolation, Co-Culture Assay, Enzyme-linked Immunosorbent Assay, cDNA Synthesis, Expressing, RNA Expression, Generated, Virus, Mutagenesis, SDS Page, Western Blot, Control, Flow Cytometry, Cell Fractionation, RNA Extraction, Derivative Assay, Positive Control, MTS Assay

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