Apoptosis assay

Apoptosis was measured in Ghost(3) cells 30 h post-infection or drug treatment using the NucViewTM 488 Caspase−3 Assay Kit (Biotium) in a 96 well format. Data was analyzed in MATLAB using a probabilistic region method to measure cell attachment and expressed as percentage cell survival. Notably, as the kit was only available with a 488nM dye, cells were analyzed at the 30 h time-point to minimize GFP input from the integrated Tat-driven reporter. An average of 3,000 cells per condition were analyzed with the exception of cells treated with Doxorubicin first followed by HIV-1 infection (Figure ​(Figure1A)1A) and cells transfected with lincRNA-p21 overexpression constructs followed by Doxorubicin treatment (Figure S2H). These conditions yielded too few attached cells (<20) at 30 h for similar analysis. Apoptosis was measured in Mφs 9 days post-infection and/or 2 days after drug treatment using the fixable viability dye Aqua Live/Dead (Thermo Fisher Scientific) in combination with the FLICA 660 caspase 3/7 dye (Thermo Fisher Scientific) after detachment using StemPro® Accutase® (Thermo Fisher Scientific). Cells were then treated with cytofix/cytoperm and perm/wash solutions (BD Biosciences) and stained with FITC-conjugated anti-p24 antibody. A minimum of 10⁴ events were acquired per treatment on a BD LSRForstessa™ flow cytometer.

HIV-1 masks DNA damage, protects against additional lethal DNA damage, and prevents lincRNA-p21 upregulation. (A) Ghost(3) cells infected 24 h prior to Doxorubicin treatment have numerous DSBs as detected by H2A.XpSer139 immunofluorescence staining (+HIV+Doxo.) but do not undergo apoptosis when normalized to infected cells (HIV 1st Doxo. 2nd; orange bars). Cells infected after exposure to Doxorubicin (+Doxo.+HIV) have extensive H2A.XpSer139 staining and do undergo apoptosis. Too few attached cells present for statistical analysis (<20). (B) Ghost(3) cells support HIV-1 replication following additional lethal DNA damage (+HIV+Doxo.) as detected by quantitative real-time RT-PCR analysis of HIV-1 Gag expression relative to the HPRT housekeeping gene and normalized to uninfected cells (mean ± SE of 3 biological replicates in triplicate). (C) The percentage of apoptotic HIV-infected Mφ (small gray wedges, upper row) does not significantly increase in HIV-infected Mφ exposed to Doxorubicin (small gray wedges, lower row) across 3 separate donors (biological replicates). (D) Mφ were infected with HIV-1 at a physiologically relevant rate as measured at 7 days by p24 flow cytometry analysis (mean ± SE of 3 biological replicates). (E) Inactive ATM dimers do not undergo autophosphorylation of serine residue 1981 in response to HIV-induced DSBs as measured by immunofluorescence staining (ATMpSer1981) in Ghost(3) cells. Phosphorylated ATM is detected in Doxorubicin-treated cells. (F) Inactive p53 monomers are not phosphorylated at serine residue 46 (specific apoptotic mark) in response to HIV-1 infection of Ghost(3) cells as measured by immunofluorescence staining (p53pSer46). Activated p53 dimers are detected in Doxorubicin-treated cells. (G) The relative nuclear fluorescence intensities (RFI) of p53ser46 residues (specific apoptotic mark) is not increased in response to HIV-1 infection of Mφ, but is increased in Doxorubicin-treated Mφ (mean ± SE of 3 biological replicates). (H) HIV-1 infection significantly decreases p53 and CDKN1A expression over time in Ghost(3) cells as detected by quantitative real-time RT-PCR analysis relative to the HPRT housekeeping gene and normalized to uninfected cells (mean ± SE of 3 biological replicates in triplicate). (I) Human lincRNA-p21, which is located upstream of CDKN1A/p21 (orange) on chromosome 6 and comprised of 2 exons (blue) and a single intron, is transcribed by p53 (yellow) in response to DNA damage. LincRNA-p21 mediates cellular apoptosis by down-regulating pro-survival genes (green) and up-regulating pro-apoptosis genes (red). (J) HIV-induced DNA damage does not lead to enhanced lincRNA-p21 expression in Ghost(3) cells as detected at 48 h by quantitative real-time RT-PCR analysis relative to the HPRT housekeeping gene and normalized to untreated cells. Doxorubicin treatment does lead to enhanced lincRNA-p21 expression (mean ± SE of 3 biological replicates in triplicate). (K) LincRNA-p21 expression is significantly decreased in HIV-infected Mφ (7 days), with an even greater decrease observed in infected Mφ exposed to Doxorubicin (HIV+Doxo. at 8 days) as detected by quantitative real-time RT-PCR analysis relative to the HPRT housekeeping gene and normalized to untreated cells (mean ± SE of 3 biological replicates in triplicate). Cells were counterstained with DAPI; scale bars = 10 μM; two-tailed paired Student T-test, ***p < 0.001, **p < 0.01, *p < 0.05, NS, not significant; see also Figure S1.