MOESM2 of Tat inhibition by didehydro-Cortistatin A promotes heterochromatin formation at the HIV-1 long terminal repeat
Creators: name: Li, Chuan
name: Mousseau, Guillaume
name: Valente, Susana
Publisher: name: figshare
Year: 2019
Subjects: subject: Cell Biology
subject: Molecular Biology
subject: Pharmacology
subject: Immunology
subject: FOS: Clinical medicine
subject: Cancer
subject: Mental Health
subject: Plant Biology
subject: FOS: Biological sciences
subject: Virology

description: Additional file 2: Figure S1. dCA inhibits HIV expression in HIV chronically infected HeLa-CD4 cells. a HIV integration level in the chronically infected HeLa-CD4 cell model. Genomic DNA extracted on the indicated days of dCA treatment was amplified by Alu-PCR followed by a nested RT-PCR. HIV proviruses were normalized to genomic GAPDH DNA. Data are the mean ± standard error. b Induction of TBP-2 expression by SAHA. The chronically infected cells grown more than 280 days in dCA and fresh HeLa-CD4 cell were activated with SAHA for 24 h. TBP-2 mRNA production was quantified from cDNAs prepared from total RNA. Results were normalized as mRNA copies per GAPDH mRNA, and data represent mean ± standard error. Results are representative of two independent experiments. c Induction of IL-1β expression by PMA/Ionomycin in the long-term-treated HeLa-CD4 cells. Cells grown more than 280 days and fresh HeLa-CD4 cells were activated with PMA/ionomycin for 24 h. IL-1β mRNA production was quantified from cDNAs prepared from total RNA. Results were normalized as mRNA copies per GAPDH mRNA, and data represent the mean ± standard error. Results are representative of two independent experiments. d Induction of histone H3 acetylation by SAHA in long-term-treated HeLa-CD4 cell. The cell samples from panel B were used for WB analysis with antibody recognizing total histone H3 or N-terminus acetylated H3. The amount of acetylated-H3 was normalized to total histone H3 and labeled below. Results are representative of two independent experiments. e PMA/ionomycin-induced viral production in HeLa-CD4 chronic infected cells. Cells treated with ART and ART + dCA (10 nM) after day 280 were stimulated with PMA/Ionomycin for 24 h. Capsid production was quantified via a p24 ELISA. Data are average of 3 independent experiments, and the error bars represent the SD of 3 independent experiments (ND, not detected). f PMA/ionomycin-induced viral mRNAs production in HeLa-CD4 chronic infected cells. Cellular samples from panel B were used for RNA extraction, and cDNAs from extracted total RNA were quantified by RT-qPCR using primers to the Nef region. Results were normalized as the number of viral mRNA copies per GAPDH mRNA. Viral mRNA generated in the ART control was set to 100%, and the error bars represent the SD of 3 independent experiments. g Distribution of RNAPII on the HIV genome treated or not with dCA. ChIP assay was performed on cells samples from panels F and G. After subtracted with the background of the isotype IgG control, the results are presented as percent immunoprecipitated DNA over input. Error bars represent the SD of 3 experiments for each primer set. h The chromatin structure of the HIV LTR in chronic infected HeLa-CD4 cell stimulated with or without PMA/ionomycin. Data are average of 3 independent experiments, and error bars represent the SD of 3 experiments for each primer set. i The recruitment of PBAF complex on HIV promoter DNA in cells stimulated with PMA/Ionomycin as determined by BAF180 ChIP. After subtracted with the background of the isotype IgG control, the results are presented as percent immunoprecipitated DNA over input. The promoter of GAPDH was used as the control. Data are average of 3 independent experiments, and error bars represent the SD of 3 experiments for each primer set. j The recruitment of BAF complex on HIV promoter DNA in cells stimulated with PMA/Ionomycin as determined by BAF250 ChIP. After subtracted with the background of the isotype IgG control, the results are presented as percent immunoprecipitated DNA over input. The promoter of GAPDH was used as the control. Data are average of 3 independent experiments, and error bars represent the SD of 3 experiments for each primer set. Statistical significance was determined using the unpaired t-test (*P &lt; 0.05, **P &lt; 0.01).