Figure 3.
Elevated expression of HO-1 is critical for HIV-1 inhibition. (A) Upregulated HO-1 levels in SCT PBMCs. PBMCs (4 HbAA, 24% female; 4 HbAS, 100% female) were lysed, and HO-1 expression was measured in lysates by using an enzyme-linked immunosorbent assay (ELISA) from 4 control and 4 SCT PBMCs. (B) Upregulated HO-1 mRNA levels in SCT PBMCs. RNA expression of HO-1 was analyzed from 4 HbAA (50% female) and 4 SCT (75% female) PBMCs. RNA was isolated by TRIzol, reverse transcribed, and analyzed by real time PCR. Primers for 18S rRNA were used as a reference for ΔΔCt analysis. (C and D) Small molecule inhibitors of HO-1 reversed HIV-1 inhibition in SCT PBMCs. SCT PBMCs were purified and activated with phytohemagglutinin and interleukin-2 and treated with 2 μM snIX before the infection with HIV-1 (IIIB). Samples were collected 4 days’ postinfection. Supernatants were used to measure p24 by using an ELISA (panel C, 6 HbAA, 50% female; 6 HbAS, 66.7% female). Cells were collected and RNA was extracted, reverse transcribed, and analyzed with primers for HIV-1 gag gene by real-time PCR using 18S RNA as a reference (panel D, 3 HbAA, 66.6% female; 3 HbAS, 66.6% female). Data are shown as means. P values were calculated by using the Student t test. (E-F) Expression of HO-1 inhibits HIV-1. In panel E, 293T cells were transfected with the indicated amounts of HO-1–expressing vector. Cell lysates were resolved on 4% to 12% Bis-Tris gel and probed by Western blotting (WB) with antibodies against c-myc and β-actin as loading control. Results were quantified by using ImageQuant Software. Bars represent quantification from 3 independent WB experiments. Data are shown as mean ± standard deviation. In panel F, HO-1–expressing cells were further infected with HIV-1-LUC-G, and luciferase activity was measured 2 days’ postinfection. Plasmid amounts are shown in calculation for a 6-well plate to be comparable with panel E. Data are shown as mean ± standard deviation (n = 3 for each sample). P values were calculated by using the Student t test.

Elevated expression of HO-1 is critical for HIV-1 inhibition. (A) Upregulated HO-1 levels in SCT PBMCs. PBMCs (4 HbAA, 24% female; 4 HbAS, 100% female) were lysed, and HO-1 expression was measured in lysates by using an enzyme-linked immunosorbent assay (ELISA) from 4 control and 4 SCT PBMCs. (B) Upregulated HO-1 mRNA levels in SCT PBMCs. RNA expression of HO-1 was analyzed from 4 HbAA (50% female) and 4 SCT (75% female) PBMCs. RNA was isolated by TRIzol, reverse transcribed, and analyzed by real time PCR. Primers for 18S rRNA were used as a reference for ΔΔCt analysis. (C and D) Small molecule inhibitors of HO-1 reversed HIV-1 inhibition in SCT PBMCs. SCT PBMCs were purified and activated with phytohemagglutinin and interleukin-2 and treated with 2 μM snIX before the infection with HIV-1 (IIIB). Samples were collected 4 days’ postinfection. Supernatants were used to measure p24 by using an ELISA (panel C, 6 HbAA, 50% female; 6 HbAS, 66.7% female). Cells were collected and RNA was extracted, reverse transcribed, and analyzed with primers for HIV-1 gag gene by real-time PCR using 18S RNA as a reference (panel D, 3 HbAA, 66.6% female; 3 HbAS, 66.6% female). Data are shown as means. P values were calculated by using the Student t test. (E-F) Expression of HO-1 inhibits HIV-1. In panel E, 293T cells were transfected with the indicated amounts of HO-1–expressing vector. Cell lysates were resolved on 4% to 12% Bis-Tris gel and probed by Western blotting (WB) with antibodies against c-myc and β-actin as loading control. Results were quantified by using ImageQuant Software. Bars represent quantification from 3 independent WB experiments. Data are shown as mean ± standard deviation. In panel F, HO-1–expressing cells were further infected with HIV-1-LUC-G, and luciferase activity was measured 2 days’ postinfection. Plasmid amounts are shown in calculation for a 6-well plate to be comparable with panel E. Data are shown as mean ± standard deviation (n = 3 for each sample). P values were calculated by using the Student t test.

Close Modal
This Feature Is Available To Subscribers Only

or Create an Account

Close Modal
Close Modal